Epstein-Barr Virus (EBV) is an ubiquitous human herpes virus that was first discovered in association with the African (endemic or e) form of Burkitt's lymphoma (BL). Subsequently the virus was also found associated with nasopharyngeal carcinoma (NPC) and was shown to be the causative agent of infectious mononucleosis (IM). Infection usually occurs during early childhood, generally resulting in a subclinical manifestation, occasionally with mild symptoms. Infection during adolescence or adulthood, however, can give rise to IM characterized by the presence of atypical lymphocytes in the periphery. The bulk of these lymphocytes are T lymphocytes; however, included in their number are a small population of B lymphocytes infected by EBV. The infection of B lymphocytes may also be accomplished in vitro. Such cells become transformed and proliferate indefinitely in culture and have been referred to as "immortalized", "latently infected" or "growth transformed". As far as is known, all individuals who become infected with EBV remain latently infected for life. This is reflected by the lifelong continuous presence of small numbers of EBV-genome positive transformed B-cells among the circulating peripheral blood lymphocytes and the continuous but periodic shedding of virus in the oropharynx.
In the vast majority of cases EBV infection results in a lymphoproliferative disease that may be temporarily debilitating, but is always benign and self-limiting. In certain immunosuppressed individuals, however, the result can be full-blown malignancy. This occurs in individuals who are immuno-suppressed intentionally, particularly children receiving organ transplants who are treated with cyclosporine A, or opportunistically, as in the case with individuals infected with HIV, or genetically, as in the case of affected males carrying the XLP (x-linked lymphoproliferative syndrome) gene. In these cases the resulting malignancies derive from the polyclonal proliferation of EBV-infected B cells. In addition, in such patients uncontrolled epithelial replication of the virus is detectable in lesions of oral hairy leukoplakia. Thus, the immune response plays a central role in the control of EBV infection.
As mentioned above EBV is a member of the herpesviruses. It possesses the following structural properties:
The EBV genome consists of a linear double stranded DNA molecule (172,000 basepairs).
The virion consists of a core (proteins and DNA), surrounded by an icosahedral capsid, and a membrane envelope enclosing the capsid. The icosahedral capsid is built up of hexameric and pentameric capsomeres. The membrane envelope consists of a protein/lipid bilayer membrane with spikes on its outer surface. The space between the capsid shell and the envelope is filled with amorphous protein, called the tegument.
Like all herpesviruses, EBV is capable of establishing a latent life-long infection in its host subsequent to primary infection. This latency represents a perfect balance between EBV and its human host, controlled by the hosts immune system.
To date most biochemical and biological studies have been performed on three prototype strains of EBV, being B95-8 (transforming virus produced in a marmoset cell line), P3HR1 (non-transforming virus produced by a Burkitt's lymphoma tumor cell line) and Raji (latent virus in a Burkitt's lymphoma tumor cell line).
During the last few years the entire DNA sequence of prototype virus strain, B95-8, has been determined. Analysis of this sequence has resulted in the identification of more than 80 open reading frames (Baer et al., 1984, Nature 310, p. 207-211).
The biology of EBV poses a special problem to investigators because its biological characteristics (latent infection) do not lend itself to the classic virus analysis. Furthermore, its cell and host range are effectively limited to human (and those of a few higher primates) B-lymphocytes and epithelial cells which are generally not amenable to culture in vitro. In addition, the absence of a fully permissive cell type, one in which the virus lytically replicates, has severely limited the ability to produce large amounts of the virus. DNA molecules of B95-8, P3HR1- and Raji-isolates have been the prototypes for detailed restriction endonuclease mapping, and for cloning into Escherichia coli (E.coli) plasmids and in bacteriophage lambda, and for nucleotide sequencing.
The EBV-genome consists of a single double stranded DNA molecule build-up with unique and tandemly repeated DNA-elements, Each end of the DNA molecule contains multiple terminal sequences which permit covalently linking and circularization of the genome. In virus particles the EBV-genome is only detectable in a linear form. On the contrary, it exists as a circular episome inside the nucleus of latently infected cells, and occasionally becomes integrated into the host cell chromosomes.
The internal repeat sequences, IR1 to IR4, separate the EBV-genome into 5 unique regions. The U2 and U3 regions vary extensively among different EBV isolates and, the former being almost entirely deleted in the P3HR-1 strain of EBV.
The nomenclature for EBV reading frames is based on their position in the virus genome. The names begins with the initials of the BamH1 or EcoR1 restriction fragment where expression begins. The third letter in the name is L or R, depending or whether the expression is leftward or rightward on the standard map. (So BLLF2 is the second leftward reading frame starting in BamH1 restriction fragment L.).
The serological classification of virus antigens in the production cycle of EBV is based on different fluorescence techniques.
Antigens specifically detected by means of the anti-complement immunofluorescence technique in the nucleus of fixed latently infected B-cells (e.g. Raji-cells) are classified as Epstein-Barr nuclear antigens (EBNA).
Upon activation of viral gene expression by chemical or viral factors a class of early antigens (EA) is detected whose synthesis is not blocked by inhibition of viral DNA synthesis. Dependent on the type of fixative used (Methanol or Acetone) two distinct sets of EA are detectable, EA.sub.R and EA.sub.D. EA is detectable by indirect immunofluorescence in the cytoplasm and nucleus of induced cells. Following onset of viral DNA-synthesis (and depending upon it) virus structural proteins (VCA) are synthesized which are detectable by indirect immunofluorescence in the cytoplasm and nucleus of virus producer cells (e.g. P.sub.3 HR.sub.1 cells). On the surface of viable infected cells, induced for virus production a set of antigens (MA) is detectable by indirect immunofluorescence. These antigens can also be found on the viral envelope and are important targets for virus neutralization.
Detection of EBV-specific antibodies in human sera can routinely be performed by serological techniques as described by Henle and Henle (Human Pathology, 5, 551-565, 1974).
Based upon biochemical and immunofluorescence data it is possible to distinguish five different classes of antigen molecules. The different viral polypeptides are designated by their molecular weight, and no common nomenclature has been established for all EBV-proteins in order to allow their unique description.
The five different groups of antigens are:
A. The group of antigens which are expressed during a state of latency (EBNAs and LMPs).
B. The group of antigens which are responsible for genome activation and initial induction of viral replication (IEA).
C. The group of antigens which are induced by IEA-gene products and which are required for replication of viral DNA; these antigens are mostly viral enzymes (EA).
D. The group of antigens which are structural components of the viral particle and are expressed late in the viral replication cycle (VCA), after initiation of viral DNA-synthesis.
E. The group of antigens which are expressed in the cell membrane of the infected cell (MA).
Epstein-Barr Early Antigens (EBV-EA)
EBV-early antigens (EA) are expressed in EBV-producer cells before onset of viral DNA-synthesis and can be studied specifically when such producer cells are treated with inhibitors of viral DNA polymerase (e.g. phosphonoacetic acid). Alternatively EA can be detected in cells abortively infected with EBV or in non-producer lymphoblastoid cells (e.g. Raji cells) activated with chemicals such as IUdR or BUdR; or TPA and butyrate.
The EA antigens represent a group of viral proteins required for both shutdown of host macromolecular synthesis and initiation of viral DNA-synthesis.
Although the exact nature of all EA-complex proteins is not known to date, some of its components have been defined molecularly in recent years.
By immunofluorescence (IF) analysis using both human sera and monoclonal antibodies two sets of Early Antigens are described which differ in their sensitivity to fixatives such as acetone and methanol. One IF-pattern is the diffuse type (D), with staining of both the nucleus and the cytoplasm, whereas the other is restricted (R) to filamentous material in the cytoplasm only. It is found that the R component is destroyed by methanol or ethanol fixation but resistant to acetone, whereas the D component is resistant to these fixatives.
The EA-D complex is composed at least of the following EBV-genome encoded proteins, P47-54 (BMRF1) the DNA-polymerase associated protein, P138 (BALF2) the major DNA binding protein, p110 (BALF5) the DNA-polymerase, p55 (BGLF5) the alkaline DNAse, P65 (BXLF1) the thymidine kinase and P52 (BMLF1) the early transactivator.
The EA-R complex is composed of at least the following EBV proteins, P85 (BORF2) the ribonucleotide reductase large subunit, p30 (BaRF1) the RR small subunit and P17 (BHRF1) the Bcl-2 homologue.
Antibodies to EBV-EA complex proteins are generally detectable in patients with active (acute or chronic) EBV-infections, with anti-EA-R being more frequently detectable in apparently healthy blood donors.
Antibodies of IgG, IgM and IgA classes to EA-D complex proteins have been detected in acute phase of mononucleosis, with IgM and IgA disappearing more rapidly than IgG during convalescence. In (severe) chronic EBV infections high titers of IgG antibodies are found to both EA-D and EA-R complexes, with occasional IgA but no IgM. In Nasopharyngeal Carcinoma high titers of both IgG and IgA are found to EA-D, the latter being of diagnostic and prognostic importance for disease monitoring. In contrast, another EBV-associated malignancy, Burkitt Lymphoma, is frequently associated with high IgG titers to the EA-R component.
In the vast majority of cases the antibody responses described above have been studied using indirect immunofluorescence techniques on different EBV cell lines induced for EA expression and fixed with acetone or methanol. More molecular defined serological studies have been started only recently.
At present, the underlying mechanism(s) of these different immune responses to EA-D and EA-R complexes is not defined, nor is it clearly defined which EA-D or EA-R proteins are detected by human antibodies in different EBV disease syndromes.
As mentioned above, some of the molecular characteristics of both EA-D and EA-R complex components have been described in some detail in recent years and their coding open reading frames have been located on the viral genome.
Production of these components from EBV-producer cells however is complex and gives low yields due to the low level of expression of these proteins in cell culture. This has prevented the development of more simple diagnostics as alternative to the laborious and subjective IF-based serological tests.
Expression of defined components in alternative host systems has been described, but their routine application in diagnostic tests requires high level of purification, in order to remove potentially interfering host proteins (e.g. E.coli).
At present EBV specific serodiagnosis is accomplished by rather subjective immunofluorescence tests. Progress to more simple and uniform diagnosis (e.g. ELISA) is hampered because bulk production and purification of viral antigens are not possible using standard virus producing cell lines.
The only way to achieve this would be to use alternatively prepared EBV antigen(s). These EBV antigens could be prepared with either genetic engineering techniques or synthetic peptide techniques.
For the development of a specific and sensitive method to enable a reliable diagnosis to be made in various phases of the infection with EBV it is of great importance to identify immuno-dominant viral proteins and epitopes thereof.